DE19514310A1 - Vectors for the transfection of eukaryotic cells, their use and thus transfected target cells - Google Patents

Abstract

The invention relates to cloning vectors for eucaryotic cells comprising an expression cassette in which the transgene is arranged behind the regulating component and the transgene is followed by an internal ribosome entry sequence and a selection gene. These vectors are suitable for the transfection of eucaryotic cells usable in gene therapy.

Description

The present invention relates to vectors that are serve non-cell DNA in eukaryotic cells, in particular Vertebrate target cells to infiltrate the on this foreign-encoded DNA as efficiently as possible express.

To get the target cells to produce a foreign gene product express, the DNA must first in the target cells be introduced. Plasmid DNA can be used for this purpose Transfection, for example by electroporation, Lipofection, calcium precipitation or particle resolution in the target cells are introduced.

Another way of getting the foreign DNA into the target cells to introduce is the use of retroviruses in which the DNA is encased in a protein. In the case of retroviruses the DNA becomes stable in the genome of dividing cells built-in.

When using plasmids, the foreign DNA is only added a small part of the cells that contain the foreign DNA have really built into the genome.  

DNA transfer using retroviruses is more efficient, but also harbors greater biological hazards.

The present invention therefore relates to vectors which are attributable to the plasmid vectors, their use and thus transfected target cells.

It is known that the transfection efficiency at Plasmid vectors leave something to be desired. Expression too of the gene encoded by the foreign cell DNA, which follows is often referred to as a transgene satisfactory. The object of the present invention is therefore, to provide vectors that have these disadvantages overcome.

The efficiency of a transfection vector is determined by its Components and the interaction of these components decisively determined. An integral part of Transfection vectors is a selection marker that it allowed the transfected cells from the not distinguish transfected cells. Mostly it is a gene whose gene product is resistant to a gene Antibiotic causes. After transfection you can only such cells grow that this gene product (resistance) produce successfully. For vertebrate cells is common uses the neomycin resistance gene ("neo").

Vectors also have regulatory sequences that the transcription and translation of the gene product, which in control of the target cell.

Vectors also have such genetic elements that are required to multiply the vector.

Finally, a transfection vector has the gene that is in the target cells should be brought to expression. This Because of its gene product, transgene is transferred to the target cell  built-in. If the transgene is for a growth factor or a cytokine or other therapeutically relevant protein the corresponding transfected cell can also be encoded gene therapy can be used.

The present invention therefore relates to Vectors for transfection of cells that express have a cassette with more than one cistron, which in Direction from the 5'-end to the 3'-end of the following components exhibit:

• a) at least one regulatory element;
• b) at least one gene to be expressed;
• c) at least one internal ribosome entry sequence (IRES);
• d) at least one selection gene.

The regulatory element (a) can comprise a promoter, which is preferably selected from the group consisting of CMV promoter and β-actin promoter. Furthermore, that regulatory element further reinforcing Have components (so-called "enhancers").

The gene (b) to be expressed is preferably genes coding for interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), Interleukin-6 (IL-6), granulocyte colony stimulating Stimulate factor (G-CSF), granulocyte-macrophage colony the factor (GM-CSF), erythropoietin (EPO), stem cell factor (SCF), tumor necrosis factor-α (TNF-α), interferon-α (IFN-α), Interferon-β (IFN-β) or interferon-γ (IFN-γ). However, it is also possible to express other genes, for example the "green fluorescent protein" or the β- Galactosidase.

It is essential that the vector according to the invention is internal Ribosome entry sequence (IRES) (c), which in  preferred embodiment is selected from the internal Ribosome entry sequences originally from Picornaviruses or the Encephalomyocarditis virus.

The vectors according to the invention have a selection gene (d) which is preferably selected from resistance genes against Antibiotics, especially the neomycin phosphotransferase gene and the hygromycin phosphotransferase gene.

In a very particularly preferred embodiment, the Vector according to the invention a selection gene, the cDNA of Herpes simplex thymidine kinase gene fused to DNA, coding for neomycin phosphotransferase or hygromycin Phosphotransferase includes. In one of these preferred The chimeric gene has the entire for the embodiments DNA encoding neomycin resistance and the entire cDNA coding for the thymidine kinase gene, these two Genes are linked by a "hinge". At this hinge is preferably a short one Amino acid sequence, particularly preferred by four Glycine residue is formed.

The vectors according to the invention also show in preferred embodiment in the direction of transcription the selection gene a gene region intron / poly AD. Of the preferably used in the vectors according to the invention Section "Intron / poly AD" comes from the SV40 and virus contains its small intron and a very efficient one Polyadenylation site. Introns are known to be small Sections in DNA and primary RNA that make up the ) interrupt dead parts of the nucleic acids. In the course of Maturation of the RNA removes these introns (splicing), see above that only the coding regions of the RNA remain.

It has been found that for efficient Expression of transgenes at least once spliced the RNA is advantageous. Therefore, in a preferred  Embodiment in the β-actin promoter another intron be present at the end of the β-actin promoter.

The polyadenylation site at the end of a transcription Unit serves to complete the RNA polymerase II activity and thus indicates the point at which the transcription admitted finish is. The polyadenylation site usually forms linked to an AATAAA sequence the position at which the so-called poly-adenosine tail attached to the immature RNA becomes. This poly-A tail is probably used for Stability of RNA and is a sign of maturity eukaryotic messenger RNA.

Finally, the vectors according to the invention in preferred embodiment at the 3'-end of the construct one NTS sequence on. In a preferred embodiment, the NTS Sequence a short murine DNA sequence with about 50 base pairs long AT-rich portions. This sequence originally came from from rDNA cistrons in the field of RNA polymerase I Start of transcription. If this sequence in plasmids is integrated and with these vectors murine cells are transfected, there is an amplification of the Vector DNA. The latter is then, in high number of copies in the genome integrated, found again. For the transfection of human cells can have corresponding human NTS sequences Find use.

The vectors according to the invention have an expression cassette with more than one cistron; are preferred dicistronic expression cassettes. Under a cistron one understands a range of DNA that is for a particular Encoded polypeptide chain. According to the invention encodes one Expression cassette for at least two polypeptides, namely the transgene and the gene product of the selection gene.

The invention also relates to eukaryotic cells which were transfected with a vector according to the invention.  

The eukaryotic cells are more preferred Embodiment around human cells, the most preferred selected from the group comprising fibroblasts, Bone marrow progenitor cells, progenitor cells of white Blood cells, Langerhans cells and dendritic Cells.

According to the invention, the cells can be used for Expression of genes in vitro, but also for the expression of Genes in vivo, preferably the transfected cells Patients are administered and the cells contain the cloned gene express in the patient.

Another use of the transfected according to the invention eukaryotic cells are used in farm animals. The vectors according to the invention can be used to eukaryotic cells, preferred by each Farm animal origin to transfect.

The starting material can preferably be from the respective animal originating fibroblasts, bone marrow progenitor cells, Precursor cells of white blood cells, Langerhans'sche Cells or dendritic cells can be used. These Cells are then with the vector according to the invention transfected. The vector according to the invention then includes this desired transgene. Depending on the purpose achieved, it can be this is a corresponding gene. It is exemplary wise conceivable, the appropriate growth hormone in this way to bring in animals that are attracted to meat production will. This enables faster growth to be achieved. Another application is for laboratory animals. A transgene can be used in laboratory animals in this way be introduced without requiring the To change the germ lines of animals (transgenic animals). It is particularly advantageous in this application if a Vector is used which has a "suicide" gene. Thereby  can express the transgene to the desired one Time can be switched off.

In Fig. 1 the difference between a preferred vector according to the invention and the classic vector type is shown schematically. In the classic vector type known from the prior art, the expression of the transgene is controlled independently of the expression of the resistance marker (here: neo), which is why two different promoters must also be used to regulate the expression.

In contrast to this, the vector according to the invention both the transgene and the selection gene into one large cassette, in which both the transgene and also the selection gene from a single promoter to be controlled.

The insertion of the IRES sequence between the transgene and the Selection marker allows translation to resume the common mRNA through the ribosomes at the end of the IRES Sequence. Usually in eukaryotic cells Translation after the stop codon in the cDNA of the transgene cancel and thus the translation of the selection marker thwart. The IRES sequence is of viral origin and forms an RNA hyperstructure identified by ribosomes as (new) Starting point of translation is recognized, so that despite the Stop codons at the end of the transgene section behind the IRES Sequence of other proteins can be translated.

Basically, it would also be possible behind or in front of that Selection gene and insert another IRES sequence this additional IRES sequence to bind another gene. This could result in additional genes in multicistronic Expression cassettes can be cloned and there could be several Proteins are brought to expression. This can especially play a role when  Gene therapy introduced several gene products into target cells and these gene products are expressed at the same time should be.

The internal ribosome entry sequence (IRES) was added to several Picorna viruses were found. The IRES causes the translation of the mRNA does not cap the RNA must be provided.

The selection gene is usually followed by an area Intron / poly AD.

In a preferred embodiment, the inventive Vectors at the 3′-end, ie at the "rear" end of the construct the so-called NTS sequence. The NTS sequence has one positive influence on the transcription of the construct Change in the local chromosome structure and can lead to Contribute amplification of the construct.

The vectors of the invention have at the 5 'end, that is "front" end, at least one regulatory element. The regulatory elements are in preferred embodiment around promoters, with particular preferably the so-called CMV promoter or the human β-actin Promoter is used. The CMV promoter is a very strong promoter, with the help of which high amounts in cell culture on transgene, for example G-CSF. In animal experiments, however, it was determined that this Promoter was quickly turned off when the one with the Vector modified cells according to the invention in mice have been transplanted.

The human β-actin which is preferably used according to the invention Promoter is used after returning the transfected cells switched off much later (more than 10 days). Compared to the CMV promoter, the β-actin promoter in human fibroblast cells, however, only about 50% of the  CNV promoter activity. Depending on that Intended use must therefore be appropriate suitable promoter can be used. In addition to that Promoters can also do other transcription-enhancing ones Elements, so-called enhancers, are installed.

A gene can be used as the selection gene Gene product causes resistance to antibiotics. In a particularly preferred embodiment of the present Invention, the selection gene is a fusion gene from the gene for thymidine kinase and the gene that is resistant to Neomyzin causes. Co-expression of the thymidine kinase gene, which is derived from herpes simplex virus, allows cells that expressing thymidine kinase by gancyclovir (GCV) selectively kill. So if the vectors of the invention used to transfect cells that To use gene therapy, it is desirable to use one Having means at hand to target these cells again kill. This is when using the invention preferably used fusion protein by the parenteral Application of Gancyclovir possible. Such "suicide" genes are particularly preferred for the vectors according to the invention used since it must be possible to be genetically manipulated Switching off cells during gene therapy. It could also be shown experimentally that the trans infected cells can be switched off again in vivo.

The vector constructs according to the invention are relatively large. It is therefore not always easy to clone the corresponding transgenes. According to the invention, therefore, further constructs were developed which only contain parts of the overall construct. As Fig. 2 shows schematically, it is possible to use only parts of the GMV promoter or the β-actin promoter and the gene to be cloned (here hu G-CSF). Such partial vectors allow a simple replacement of the gene to be expressed (here hu G-CSF) by a desired new sequence, which can then be cloned into the overall construct via two singular interfaces (shown here as E, B). This can be demonstrated without problems using the β-galactosidase gene.

Fig. 3 shows another vector in which the gene to be expressed is the marker gene GFP (Green fluorescent protein). The peculiarity of this vector can be seen in the fact that the vector has unique interfaces for restriction endonucleases, namely PinA I and BamH I, via which other transgenes can be cloned, so that this vector can be used excellently in practice. A quick exchange of promoters is also possible with this vector. The use of the unique restriction site PinA I has the advantage that it is compatible with DNA ends which were generated by the restriction endonucleases Xma I, SgrA I, Eco56 I, Cfr101 or BseAI. BamH I is compatible with fragments generated by Bgl II or Bcl I. With the use of GFP in the vector, it is possible to conveniently test whether the vector is suitable for the intended purposes.

The effectiveness of the vectors according to the invention was checked in the examples below. In the following examples, different types of vectors were compared. When testing G-CSF production, the same vector types were compared once with and once without an NTS sequence. The classic vector (with two promoters) was used for the values shown in FIG. 6, whereas FIG. 7 shows results which were obtained with a vector according to the invention.

The following examples show the influence the NTS sequence can be shown to have a singular effect e.g. B. only to exclude the CMV promoter. Farther were vectors according to the invention with conventional vector  compared constructions from which it follows that the vector according to the invention forms significantly more G-CSF.

example 1 Transfection efficiency

The first step in testing a transfection vector consists of examining to what extent and in what goodness the vector its information in the target cell can bring. The determination of the Serve transfection efficiency, d. H. how many target cells contain the vector after expression and express (at least) the marker gene. In the present invention the neomycin resistance gene was used as the selection gene.

Generally speaking, retroviruses are usually show a very good efficiency, which means that up to 100% of all cells can be reached. In contrast, with plasmids only one in 1,000 to 100,000 cells is transfected. With plasmid vectors in particular, there is therefore an improvement in Transfection efficiency is crucial.

Human fibroblasts from the KMST-6 line were used in the present experiment. In parallel, they were transfected once with the vectors according to the invention without NTS and once with the vectors according to the invention with an NTS sequence. The experiments were carried out by lipofection. Then, under neomycin selection, it was determined how many independent colonies in cell culture grew from the original cells. Since shortly after lipofection a large number of colonies are formed in which the plasmid has not been stably integrated into the chromosome (transient transfection), the number of colonies may only be calculated after a long period of time. In the present example, the colonies were counted 14 days after the start of selection if the colonies consisted of more than 20 cells. Usually, these colonies then stably integrated the plasmid into the chromosome and they no longer die even under continued selection pressure with neomycin. The determination of the transfection efficiency is only a relative parameter because there are no standards for the selection pressure. It is therefore obvious that cells that synthesize high amounts of resistance gene product can survive even with higher amounts of inhibitors than cells that produce little or no resistance gene product. In a heterogeneous culture there will always be cells with different levels of resistance, so that the transfection efficiency can only be compared at an inhibitor concentration in the same cell type. The differences in the resistance level can also be used for an efficient selection.

In the present example, 10⁵ KMST-6 cells were lipofected with 4 µg DNA and then divided. Only then was the selection started with neomycin, so that at least the initial conditions were the same for all cells. The results of the tests are shown in Figs. 4 and 5.

It can be seen from these results that the NTS-containing vectors generally have a higher transfection efficiency than their NTS-less counterparts. After transfection, the NTS-containing plasmids lead to more colonies in each experiment in each experiment than the NTS-free plasmids. This "more" becomes more important the higher the selection pressure by neomycin is. The experiments shown in Fig. 4 were carried out with the CMV promoter. The relative improvement in efficiency is between 1.5 and 7 times.

Example 2

The experiments shown in Fig. 5 were carried out with the β-actin promoter. The relative improvement in efficiency is between 2 and 4 times. As expected, the apparent efficiencies with increasing selection pressure are of course significantly lower than with the standard value of 500 µg / ml neomycin. The difference in efficiency between NTS-containing and NTS-free vectors is statistically significant. It should be noted that the IRES construct when selected with 500 µg / ml neomycin enables a significantly higher transfection efficiency than the classic construct (160 versus 260 colonies; both values with NTS). Without NTS there is no difference.

Example 3 Production of the transgene (here: G-CSF)

In practice, it's not just the transfection efficiency significant, much more important is the high expression of the Transgenic encoded protein. In this experiment, the clones generated according to Example 1 and 2 used. about well-isolated colonies became small plastic cylinders inside out, the cells lying in the cylinder isolated and in new, separate culture vessels implemented. These clones were so far expanded until it has an area of about 1 cm² overgrow. A 24-hole plate is used here. While As the cells grow, they continuously secrete the transgene (here: G-CSF) into the culture medium, in which it is then detected can be. The culture medium becomes complete for detection removed from the cells and replaced with fresh medium. After a certain period of time, here 24 hours, it will Medium for the G-CSF determination removed and then in the ELISA checked for the cytokine content.

In order to obtain the highest possible expression of the transgene, the transfected KMST-6 cells in different Neomyzin concentrations selected. The use of high Concentrations (up to 3 mg / ml neomycin sulfate) should be the Cloning that could build higher neo-resistance was better Guarantee survival. In the case of the double RNA plasmid classic vector is said to increase G-CSF production  appear as a side effect (high neomycin resistance only with multiple copies, especially when installing the plasmid favorable places in the chromosome or - at NTS - local Activation of the chromosome after installation of the plasmid). At the construct according to the invention should be based on the selection high neomycin resistance at the same time on high production on transgene (here: G-CSF) because they are the same RNA yes carries both information.

The results of this experiment are summarized in Figs. 6 and 7. Simply increasing the neomycin concentration when selecting the clones did not result in an increase in G-CSF production. The values in columns G500 to G3000 ( Fig. 6) apply to the double RNA plasmid and the values βact500 to βact2000 ( Fig. 7) for the IRES construct according to the invention.

Fig. 6 shows results that were obtained with a classic RNA vector. The abbreviation G means without an NTS sequence, whereas NG means: classic double RNA vector with an NTS sequence. In the graphic above, G 500 means: transfection with classic vector ( 2 promoters) without NTS sequence, selection with 500 µg / ml neomycin, CMV promoter. The abbreviation NG 500 means transfection with classic vector ( 2 promoters) with NTS sequence, selection with 500 µg / ml neomycin, CMV promoter. The higher numbers 1000, 2000 and 3000 mean selection with 1000, 2000 and 3000 µg / ml neomycin.

Fig. 7 shows results which were achieved with the vector type according to the invention. The abbreviation βact 500 means transfection with the vector according to the invention without an NTS sequence, selection with 500 μg / ml neomycin, β-actin promoter. The abbreviation β + NTS 500 means transfection with vector according to the invention with NTS sequence, selection with 500 μg / ml neomycin, β-actin promoter. The numbers 1000 and 2000 indicate the selection with 1000 and 2000 µg / ml neomycin.

The number n = 16 etc. refers to the amount of examination ten clones per experiment and confirms the informative value of the Try it.

If the vectors had the NTS sequence, a significant increase in production with increasing amount of neomycin was found. In Fig. 6 the corresponding vectors are shown in columns NG500 to NG3000 and in Fig. 7 in columns β + NTS500 to β + NTS2000. The NTS sequence appears to have a direct impact on transcription. The effect of increasing the number of copies can almost be ruled out, since all very well-producing clones have only one or two copies per genome. This is less than the average number of copies measured on mixed cultures from many colonies containing 7 to 10 copies per genome for the double RNA vector or 2 to 3 copies for the IRES vector. Since the increase in the concentration of neomycin alone has no positive effect on transgene production during the selection, an explanation could be seen in the fact that the NTS sequence brings about a local improvement in the chromosome structure.

The two Figs. 6 and 7 clearly show that the vector according to the invention allows a significantly higher G-CSF production than the vector known from the prior art. While the vector of the classic type without NTS produces on average only about 5 ng / 24 hours and 24-hole plate, the mean value for the vector according to the invention is 50 ng. NTS and high neomycin selection pressure increase the production in the vector according to the invention to over 100 ng.

At the single clone level, the classic double RNA vector maximum production of 100 ng / 24 hours and  24-hole plate, but the IRES vector up to 450 ng G-CSF. It is not taken into account here that the β-actin promoter, as used in the vector according to the invention, only about 50% Has activity of the CMV promoter.

Example 4

In order to detect the expression of a transgene in the vectors according to the invention, various vectors were constructed, which are shown schematically in FIG. 8. The abbreviations used there have the following meanings: CMV = former promoter of CMV; IL-2 = cDNA for human interleukin-2; G-CSF = cDNA stimulating factor for human granulocyte colony; Neo ^r = cDNA for neomycin phosphotransferase; TK = cDNA for thymidine kinase from herpes simplex virus; IRES = internal ribosome entry sequence (from encephalomyocarditis virus); poly A = small intron and polyadenylation signal from virus SV40; Kinker = "hinge". The vector pNeoCMVIL2.3 is a classic vector with two promoters. The other vector constructs shown in FIG. 8 represent embodiments of the vector according to the invention. Either interleukin-2 or G-CSF was used as the transgene.

In a transfection experiment, Balb3T3 cells were examined cationic lipofection with the plasmids pNeoCMVIL2.3, pCMV.IL2.iresNEO and pCMV.NEO.ireslL2 transfected. All Plasmids were pre-transfected by digestion with the Restriction enzyme ScaI has been linearized. With the three Plasmids could make no difference in transfection efficiency can be observed. From any transfection approach 15 clones that were stably transfected were randomly selected Resistance to G 418 (1 mg G 418 per ml Growth medium). These clones were selected for IL-2 examined. It was found that with the clones that are associated with Plasmid pCMV.IL2.iresNEO were transfected for expression of 348 (± 293) international units IL-2 per 10⁶  Cell clones and 24 hours was found, whereas the IL-2 production of clones transfected with pNeoCMVIL2.3 (conventional vector) was significantly lower (197 ± 299 international units / 10⁶ clones × 24 hours).

Another important result of this attempt is that everyone clones transfected with vectors according to the invention which were neomycin resistant, high concentrations of IL-2 produced, whereas three clones with conventional Vector were transfected, no IL-2 was produced. However, these clones have neomycin resistance, since they otherwise could not have grown under the selection conditions.

Example 5

To check the in vivo expression of transgenes with the help of the vectors according to the invention, human G-CSF was selected, since the determination of leukocyte numbers in the peripheral blood of mice allows a simple determination of the activity of the transgene in vivo. In the experiments, highly aggressive mouse CMS-5 fibrosarcoma cells were transfected with the vectors pCMV.GCSF.iresNEO, pCMV.GCSF.iresTK / NEO and pCMV.GCSF.iresNEO / TK. Since the latter plasmid mediated only a slight resistance to G 418, it was not used further in the experiments. The following average secretion of G-CSF was determined for the transfected cells:
For cells transfected with pCMV.GCSF.iresNEO: 1.2 (± 1.5) mg / 10⁶ cells × 24 hours and
with pCMV.GCSF.iresTK / NEO: 0.37 (± 0.12) µg / 10⁶ cells × 24 hours. A significant inhibition of the growth of the latter cells (with thymidine kinase gene) was observed by adding Gancyclovir, whereas there was hardly any inhibition in the cells transfected with pCMV.GCSF.iresNEO.

To determine the function of the chimeric selection gene in vivo were tested, the cells were transfected as described above Balb / c strain mice injected. Each time 2.5 × 10⁵ cells injected into 6 mice.

Seven days after the transfected tumor cells were injected, three mice were treated by each test group 2 × intraperitoneally daily with 15 mg ganciclovir per kg body weight for 18 days each. The tumor growth and the number of leukocytes in the peripheral blood were measured in all animals.

The group that received cells with the vector pCMV.GCSF.iresNEo had been transfected (i.e. without Thymidine kinase gene) developed tumors with one exception and these animals had to be killed after 2 weeks. All the Animals from this group have GCSF-secreting tumors who did not undergo gancyclovir treatment regressed, showed exponentially increasing leukocytes numbers.

In contrast, all tumors expressing the TK / Neo ^r fusion protein completely regressed under gancyclovir treatment, with the leukocyte count also returning to the initial value.

Claims (14)

1. Vector for the transfection of cells, characterized in that it has an expression cassette with more than one cistron, which has the following components in the direction from the 5'-end to the 3'-end:

• a) at least one regulatory element;
• b) a gene to be expressed;
• c) an internal ribosome entry sequence (IRES);
• d) a selection gene.

2. Vector according to claim 1, characterized in that the regulatory element comprises a promoter selected can be from the group consisting of CMV promoter and β- Actin promoter and possibly other reinforcing Has components. 3. Vector according to one of claims 1 or 2, characterized characterized in that the gene to be expressed is selected from genes coding for interleukin-1 (IL-1), Interleukin-2 (IL-2), Interleukin-3 (IL-3), Interleukin-4 (IL-4), interleukin-6 (IL-6), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage Colony stimulating factor (GM-CSF), stem cell factor (SCF), erythropoietin (EPO), tumor necrosis factor-α (TNF-α), Interferon-α (IFN-α), interferon-β (IFN-β) or interferon-γ (IFN-γ). 4. Vector according to one of claims 1 to 3, characterized characterized that the internal ribosome entry sequence (IRES) is selected from the internal  Ribosome entry sequences originally from Picornaviruses or the Encephalomyocarditis virus. 5. Vector according to one of claims 1 to 4, characterized characterized in that the selection gene is selected from Resistance genes against antibiotics, especially neomycin Phosphotransferase gene and the hygromycin phosphotransferase Gene. 6. Vector according to one of claims 1 to 5, characterized characterized in that the selection gene comprises cDNA of the Herpes simplex thymidine kinase gene fused to DNA, coding for neomycin phosphotransferase or hygromycin Phosphotransferase. 7. Vector according to one of the preceding claims, characterized characterized in that the vector in the direction of transcription the selection gene has an intron / poly AD gene region. 8. Vector according to one of the preceding claims, characterized characterized in that the vector at the 3'-end of the construct has an NTS sequence. 9. Vector according to one of the preceding claims, characterized characterized in that the expression cassette two cistrons having. 10. Eukaryotic cell, characterized in that it has transfected a vector according to any one of claims 1 to 9 has been. 11. Eukaryotic cell according to claim 10, characterized characterized that it is a human cell, selected from the group comprising fibroblasts, Bone marrow stem cells, progenitor cells of white Blood cells, Langerhans cells and dendritic Cells.   12. Use of cells according to one of claims 10 or 11 for expression of genes in vitro. 13. Use of cells according to one of claims 10 or 11 for expression of genes in vivo, the transfected Cells are administered to patients and the cells express the cloned gene in the patient. 14. Use of cells according to claim 10 for expression of genes in vivo, the cells being administered to farm animals and the cells the cloned gene in the farm animals express.